In order to investigate the role of the platelet P2Y1 receptor in several aspects of platelet activation and thrombosis, transgenic (TG) mice overexpressing this receptor specifically in the megakaryocytic/platelet lineage were generated using the promoter of the tissue-specific platelet factor 4 gene. Studies of the saturation binding of [33P]2MeSADP in the presence or absence of the selective P2Y1 antagonist MRS2179 indicated that wild-type (WT) mouse platelets bore 150 +/- 31 P2Y1 receptors and TG platelets 276 +/- 34, representing an 84% increase in P2Y1 receptor density. This led to a well defined phenotype of platelet hyper-reactivity in vitro, as shown by increased aggregations in response to adenosine 5'-diphosphate (ADP) and low concentration of collagen in TG as compared with WT platelets. Moreover, overexpression of the P2Y1 receptor enabled ADP to induce granule secretion, unlike in WT platelets, which suggests that the level of P2Y1 expression is critical for this event. Our results further suggest that the weak responses of normal platelets to ADP are due to a limited number of P2Y1 receptors rather than to activation of a specific transduction pathway. TG mice displayed a shortened bleeding time and an increased sensitivity to in vivo platelet aggregation induced by infusion of a mixture of collagen and epinephrine. Overall, these findings emphasize the importance of the P2Y1 receptor in hemostasis and thrombosis and suggest that variable expression levels of this receptor on platelets might play a role in thrombotic states in human, which remains to be assessed.